Positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs) are powerful molecular tools to identify enzyme substrate and potent inhibitory sequences and also to provide crucial information about active site determinants. PS-SPCLs have been surveyed for furin, proprotein convertase (PC)2, PC1/3, and PACE4 and proven efficient to identify potent peptidyl inhibitors in the low nanomolar range for furin and PC1/3. We report herein the screenings of nonamidated and acetylated hexapeptide PS-SPCLs for PC5/6A and PC7. The L-configuration library surveys distinctively revealed that L-Arg, L-Lys, and sometimes L-His in all six positions would generate the most potent inhibitors for both enzymes. Based on this clear polybasic preference, L-poly-Arg peptides ranging from four to nine residues were assayed. Inhibitory potency of these polybasic peptides increased with chain length, making nona-l-Arg a potent nanomolar inhibitor of PC5/6A and PC7 (Ki of 150 and 120 nM). PC5/6 and PC7 inhibition by nona-l-arginine was equivalent to that of furin (Ki of 114 nM) (J Biol Chem 275: 36741-36749, 2000). Nona-d-arginine was a more potent inhibitor of PC5/6 and PC7 than its levorotatory version (Ki of 19 and 81 nM), reminiscent of furin (Ki of 1.3 nM) (J Biol Chem 279:36788-36794, 2004). Our data indicate that certain poly-arginine peptides represent potent inhibitors targeting PCs of the constitutive secretory pathway (furin, PC5/6, and PC7). We conclude that basic residues within PC peptide inhibitors might be responsible for targeting PCs in general and for inhibitory potency, but that select amino acid changes will be necessary to acquire true specificity toward a single PC.